The long-term goal is to contribute to an understanding of the mechanism of human cataract so that medical treatment strategies can be developed. High calcium levels are almost always detected in human cortical cataract indicating impairment of the lens pump-leak system for calcium balance. We have hypothesized that loss of calcium homeostasis might lead to a series of events contributing to cataract development. The objective of the present study is to fully describe the major mechanisms responsible for calcium homeostasis, the active outward calcium pump and the inward passive leak. We will describe the distribution and types of Ca-ATPase (pump) in the bovine and human lens and explore its susceptibility to damage by cataract-related factors such as oxidative damage and alcohol. We will also explain the dependence of Ca-ATPase function upon its lipid environment in the cell membrane. The parameter of membrane permeability (leak) to calcium will be characterized in terms of its dependence upon membrane lip id composition and alterations induced by cataract-related factors. These studies will define the precise mechanism of lens calcium homeostasis and will help to pinpoint why the lens pump-leak system for calcium is impaired in many forms of cataract. The distribution of Ca-ATPase will be determined using Western blot techniques and Ca-ATPase activity. Distribution will be determine in normal human lenses of different age and in cataractous lenses. Purified lens Ca-ATPase will be reconstituted into lens lipid vesicles to measure the parameters of calcium transport. Modulation of calcium transport by a number of cellular factors will be explored. The impact of oxidative damage on calcium homeostasis will be determined by examining Ca-ATPase kinetics and calcium transport directly. Using the lipid vesicle technology we will also correlate Ca-ATPase function with altered lipid membrane environment. Changes in lens membrane fluidity will be measured by spectroscopy. Lens membrane permeability to calcium will be measured using vesicle and liposome technology to examine directly passive fluxes using both an isotopic method and a fluorescent dye technique. Alterations in membrane permeability to calcium will be studied following alteration of lipid composition and exposure of the vesicles to cataract- related factors.